Automated Force Volume Image Processing for Biological Samples

Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image

Effects of nanoscale film thickness on apparent stiffness of and cell-mediated strains in polymers

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2006.Includes bibliographical references (leaves 65-70).The mechanical properties of compliant materials such as polymeric films and biological membranes that are of nanoscale in thickness are increasingly extracted from scanning probe microscope-enabled nanoindentation. These films are applied in various fields that require multiaxial loading conditions. The Hertzian contact models developed for linear elastic materials of semi-infinite thickness fail to accurately predict the elastic modulus E for these compliant materials. This makes it necessary to understand the evolution of stress and strain fields of these nanoscale structures. In this thesis we employ computational simulations that are based on experimental parameters for contact based analysis of compliant polymer thin films, to decouple the effect of thickness and angle of indentation on calculated mechanical properties. Traction applied by living cells to these compliant films are studied in detail. We thus identify the range of strains and material thickness for which contact models could be used to accurately predict the elastic stiffness of these polymeric films of nanoscale (<100 nm) thickness using scanning probe microscope-enabled experiments, and the volumes over which adhered cells deform these films. The key results of this thesis enable accurate experimental analysis of polymeric thin film elastic properties, and design of synthetic polymeric substrata that will dominate the mechanical environment of adhered cells.by Binu K. Oommen.S.M

Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces

Biomechanics of single cortical neurons

This study presents experimental results and computational analysis of the large strain dynamic behavior of single neurons in vitro with the objective of formulating a novel quantitative framework for the biomechanics of cortical neurons. Relying on the atomic force microscopy (AFM) technique, novel testing protocols are developed to enable the characterization of neural soma deformability over a range of indentation rates spanning three orders of magnitude, 10, 1, and 0.1 μm s[superscript −1]. Modified spherical AFM probes were utilized to compress the cell bodies of neonatal rat cortical neurons in load, unload, reload and relaxation conditions. The cell response showed marked hysteretic features, strong non-linearities, and substantial time/rate dependencies. The rheological data were complemented with geometrical measurements of cell body morphology, i.e. cross-diameter and height estimates. A constitutive model, validated by the present experiments, is proposed to quantify the mechanical behavior of cortical neurons. The model aimed to correlate empirical findings with measurable degrees of (hyper)elastic resilience and viscosity at the cell level. The proposed formulation, predicated upon previous constitutive model developments undertaken at the cortical tissue level, was implemented in a three-dimensional finite element framework. The simulated cell response was calibrated to the experimental measurements under the selected test conditions, providing a novel single cell model that could form the basis for further refinements.Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (DAAD-19-02-D-002)Joint Improvised Explosive Device Defeat Organization (U.S.) (W911NF-07-1-0035)National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Molecular, Cell, and Tissue Biomechanics Training Grant)Ecole des ponts et chaussees (France)Computation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology Allianc

COMPUTATIONAL APPROACHES TO UNDERSTAND PHENOTYPIC STRUCTURE AND CONSTITUTIVE MECHANICS RELATIONSHIPS OF SINGLE CELLS

The goal of this work is to better understand the relationship between the structure and function of biological cells by simulating their nonlinear mechanical behavior under static and dynamic loading using image structure-based finite element modeling (FEM). Vascular smooth muscle cells (VSMCs) are chosen for this study due to the strong correlation of the geometric arrangement of their structural components on their mechanical behavior and the implications of that behavior on diseases such as atherosclerosis. VSMCs are modeled here using a linear elastic material model together with truss elements, which simulate the cytoskeletal fiber network that provides the cells with much of their internal structural support. Geometric characterization of single VSMCs of two physiologically relevant phenotypes in 2D cell culture is achieved using confocal microscopy in conjunction with novel image processing techniques. These computer vision techniques use image segmentation, 2D frequency analysis, and linear programming approaches to create representative 3D model structures consisting of the cell nucleus, cytoplasm, and actin stress fiber network of each cell. These structures are then imported into MSC Patran for structural analysis with Marc. Mechanical characterization is achieved using atomic force microscopy (AFM) indentation. Material properties for each VSMC model are input based on values individually obtained through experimentation, and the results of each model are compared against those experimental values. This study is believed to be a significant step towards the viability of finite element models in the field of cellular mechanics because the geometries of the cells in the model are based on confocal microscopy images of actual cells and thus, the results of the model can be compared against experimental data for those same cells

Multiscale mechanical analysis of the elastic modulus of skin

The mechanical properties of the skin determine tissue function and regulate dermal cell behavior. Yet measuring these properties remains challenging, as evidenced by the large range of elastic moduli reported in the literature-from below one kPa to hundreds of MPa. Here, we reconcile these disparate results by dedicated experiments at both tissue and cellular length scales and by computational models considering the multiscale and multiphasic tissue structure. At the macroscopic tissue length scale, the collective behavior of the collagen fiber network under tension provides functional tissue stiffness, and its properties determine the corresponding elastic modulus (100-200 kPa). The compliant microscale environment (0.1-10 kPa), probed by atomic force microscopy, arises from the ground matrix without engaging the collagen fiber network. Our analysis indicates that indentation-based elasticity measurements, although probing tissue properties at the cell-relevant length scale, do not assess the deformation mechanisms activated by dermal cells when exerting traction forces on the extracellular matrix. Using dermal-equivalent collagen hydrogels, we demonstrate that indentation measurements of tissue stiffness do not correlate with the behavior of embedded dermal fibroblasts. These results provide a deeper understanding of tissue mechanics across length scales with important implications for skin mechanobiology and tissue engineering. STATEMENT OF SIGNIFICANCE: Measuring the mechanical properties of the skin is essential for understanding dermal cell mechanobiology and designing tissue-engineered skin substitutes. However, previous results reported for the elastic modulus of skin vary by six orders of magnitude. We show that two distinct deformation mechanisms, related to the tension-compression nonlinearity of the collagen fiber network, can explain the large variations in elastic moduli. Furthermore, we show that microscale indentation, which is frequently used to assess the stiffness perceived by cells, fails to engage the fiber network, and therefore cannot predict the behavior of dermal fibroblasts in stiffness-tunable fibrous hydrogels. This has important implications for how to measure and interpret the mechanical properties of soft tissues across length scales

Modeling and simulation in tribology across scales: An overview

This review summarizes recent advances in the area of tribology based on the outcome of a Lorentz Center workshop surveying various physical, chemical and mechanical phenomena across scales. Among the main themes discussed were those of rough surface representations, the breakdown of continuum theories at the nano- and micro-scales, as well as multiscale and multiphysics aspects for analytical and computational models relevant to applications spanning a variety of sectors, from automotive to biotribology and nanotechnology. Significant effort is still required to account for complementary nonlinear effects of plasticity, adhesion, friction, wear, lubrication and surface chemistry in tribological models. For each topic, we propose some research directions

Membrane mechanics governs cell mechanics in epithelial cell: how surface area regulation ensures tension homeostasis

Die Plasmamembranspannung von eukaryotischen Zellen soll maßgeblich zur Regulation von zellulären Prozessen wie der Zellmigration, Mitose, Endo- und Exozytose, Membranreparatur, Osmoregulierung und Zellspreiten beitragen, welche zu einer Veränderung der Membranfläche und ihrer Deformation führt. In dieser Arbeit wurde die epitheliale Zelllinie MDCK II (Madin-Darby Canine Kidney) benutzt, um spannungsgesteuerte Oberflächenregulierung zu untersuchen. Indentationsexperimente kombiniert mit dem Herausziehen von Membrannanoröhren wurden mit Hilfe des Rasterkraftmikroskops (Atomic Force Microskope, AFM) durchgeführt, um lokale Variationen in der Membranspannung und überschüssiger Membranfläche als Funktion von äußeren Reizen abzuschätzen. Die verwendeten externen Stimuli beinhalten eine Veränderung der Funktionalität des Actomyosin-Cortexes durch die Wirkung von Blebbistatin und Cytochalasin D, sowie die Manipulation der Zytoskelett-Membran Adhäsionspunkte durch Einzel-Mikroinjektion. Die Injektion von Neomycin verhindert die Anbindung von ERM-Proteinen an das Lipid Phosphatidylinositol-(4,5)-bisphosphat (PIP2) und bewirkt somit die Abkopplung des Zytoskeletts von der Plasmamembran. Als Gegenexperiment diente die Injektion des Lipids PIP2 selbst, welches zur Erhöhung der Anzahl der Zytoskelett-Membran Adhäsionspunkte führte. Weiterhin wurden die als Membranreservoire dienenden Mikrovilli durch den Entzug von Cholesterol entfernt. Auswirkung auf das Vorhandensein von Membranreservoiren hat ebenfalls die Veränderung des osmotischen Drucks innerhalb der Zellen. Zusätzlich wurden die elastischen Eigenschaften von apikalen Zellmembran-Fragmenten von konfluenten MDCK II Zellen untersucht, welche Aufschluss über die intrinsischen Membraneigenschaften ohne den Einfluss des Zytosols und Zytoskeletts geben konnten. Abschließend wurde die Mechanik von adhärierenden und spreitenden Zellen untersucht. Zusammenfassend kann gesagt werden, dass die Plasmamembran, bestehend aus einer Phospholipiddoppelschicht, lateral schwer ausdehnbar ist aufgrund ihrer flüssig-kristallinen Natur. Durch das Vorhandensein von dynamischen Membranreservoiren wie Mikrovilli, die schnell auf Veränderungen der Membranspannung durch Membranhomöostase reagieren, werden zellulare Prozesse wie die Zellmotilität oder die Anpassung an osmotischen Stress ermöglicht. In der vorliegenden Arbeit gelang es gleichzeitig, die Membranspannung und die Verfügbarkeit von Membranfläche von adhärenten konfluenten als auch von adhärierenden und spreiten Zellen zu messen. Die durchgeführten Experimente ergaben ein detailliertes Bild wie sich die zelluläre Oberflächenregulierung in der Membranmechanik widerspiegelt

CHARACTERIZATION OF VASCULAR SMOOTH MUSCLE CELL MECHANICAL AND FRICTIONAL PROPERTIES USING ATOMIC FORCE MICROSCOPY

A working hypothesis within the Laboratory of Vascular Research is that mechanical loading on vascular smooth muscle cells (VSMCs), especially due to solid contact from endovascular devices, contributes to the development of restenosis. In order to better understand the role of mechanical loading on VSMCs in vascular disease development, it is imperative to understand the mechanical properties of VSMCs themselves. To measure the viscoelastic and frictional properties of living VSMCs in an in vitro setting, an atomic force microscope (AFM) was utilized, thereby allowing for mechanical testing of living cells in a fluid environment. In the first phase of research, it was found that proliferative VSMCs, similar to those commonly found in atherosclerotic lesions, had lower stiffness and higher hysteresis values than quiescent VSMCs. Furthermore, measured stiffness values did not appear to deviate greatly within the central region of adherent cells. As VSMCs are viscoelastic, rather than purely elastic in their mechanical behavior, phase two involved the development of an AFM-based stress relaxation technique, in order to quantify VSMC viscoelastic behavior. Suitable mechanical models, including the QLV reduced relaxation function and a simple power-law model, were identified and applied to accurately describe VSMC stress relaxation. In addition, the roles of two major cytoskeletal components, actin and microtubules, in governing stress relaxation behavior, were quantified via the aforementioned mechanical models. In phase three, the surface frictional properties of VSMCs were focused upon, and a novel method to quantify surface shear forces on VSMCs using lateral force microscopy was developed. It was determined that VSMC frictional properties are greatly influenced by cell stiffness, and elastohydrodynamic lubrication was proposed as a possible cellular lubricating mechanism. During research phase four, each of the techniques developed during the preceding phases was employed to test the effects of a clinically relevant biomolecule, oxidized low-density lipoprotein (oxLDL) on VSMC mechanical properties. It was concluded that oxLDL is associated with decreased cell stiffness, and decreased viscosity, as measured by stress relaxation and indentation tests. Furthermore, frictional coefficients were found to correlate positively with more fluid-like cells. This research project has led to a better understanding of VSMC mechanical behavior, as well as the development of AFM-based techniques and models that will be useful in determining cellular mechanical and frictional effects of various stimuli in an in vitro environment